Continuous cell line and vaccine against avian coccidia

ABSTRACT

A non-lymphoid continuous cell line adapted for propagation of avian coccidia is provided. This cell line is useful for the production of vaccine antigenes for prophylactic treatment of poultry, particularly in a novel vaccine for coccidia.

This is a division, of application Ser. No. 08/182,004, filed Jul. 11,1994, now U.S. Pat. No. 5,674,484 which is the U.S. national stage ofPCT/US92/05797, filed Jul. 10, 1992, which is a continuation-in-part ofapplication Ser. No. 07/729,256, filed Jul. 12, 1991, now abandoned.

FIELD OF THE INVENTION

The present invention relates generally to a virus-free continuous cellline adapted for propagation of avian coccidia. More particularly, theinvention relates to the use of the cell line for the production ofvaccine antigens for prophylactic treatment of poultry againstcoccidiosis.

BACKGROUND OF THE INVENTION

Coccidiosis is an enteric disease of domestic and wild livestock causingacute morbidity resulting in decreased growth and feed utilization. Theavian coccidia (Genus Eimeria) are obligate intracellular protozoanparasites of the intestinal epithelium. These parasites have amonoxenous life cycle and exhibit a high degree of host-species andtissue specificity. For poultry, coccidial infection results in economicloss from stunting and skin discoloration. Overall, the combination oflosses due to coccidiosis and prophylactic medication results in a costto the poultry industry in excess of $300 million annually Danforth andAugustine, Animal Nutrition and Health, pp. 18-21 (August 1985)!.

Thousands of coccidian oocysts may be ingested at one time by a singlehost. Once ingested the parasites invade specific intestinal cells wherethey may undergo several rounds of asexual replication followed bygametogeny before millions of new parasites are shed in the litter tocomplete the life cycle. Different species of poultry suffer frominfections caused by different coccidia species. The domestic fowl(Gallus domesticus) can be infected by any of the coccidia Eimeriatenella, E. necatrix, E. brunetti, E. maxima, E. acervulina and E.praecox. The following coccidia are implicated in infections of turkeys(Meleagris): Eimeria meloagrimitis, E. dispersa, E. meleagridis, E.gallopavonis, E. adenoides, E. innocua and E. subrotunda. Domestic ducks(Anas) suffer from infections caused by Tyzzeria perniciosa and also, itis believed, by Eimeria anatis which they can acquire from wild ducks(Anas platyrhyncos). Geese (Anser) can suffer from infections caused byEimeria anseris, E. nocens and E. parvula, and in addition it isbelieved that domestic geese can acquire infections from Canada geesecaused by Eimeria hermani, E. striata and E. fulva.

Immunity to coccidiosis is reported to be highly species-specific and amanifestation of cell-mediated processes M. E. Rose, in "Biology of theCoccidia", P. L. Long, ed., University Park Press, Baltimore, pp.328-372(1982)!. Natural exposure to Eimerian oocysts elicits completeprotective immunity; this response appears to result primarily from thedevelopment of intracellular parasite stages instead of extracellularsporozoites or merozoites M. Jenkins et al, Infec. Immun., 59:4042-4048(1991)!. Although a few oocysts may confer protection to subsequentchallenge, this primary exposure impacts adversely on weight gain, feedutilization and skin pigment retention.

Current methods of control involve primarily chemotherapeutic treatmentwith anticoccidial drugs mixed into the feed. Effective compounds haveincluded sulphonamides, quinolines and polyether ionophorous antibioticsSee, e.g., L. R. McDougald, in "Biology of the Coccidia", pp. 373-427(1982)!. These compounds appear to affect parasite development atdifferent stages of their lifecycle. Over time, however, drug resistantstrains of parasites have evolved, thus severely limiting a drug'susefulness T. K. Jeffers, Avian Dis., 18:74 (1974); T. K. Jeffers, AvianDis., 18:331 (1974); and H. D. Chapman, Vet. Parasit., 15:11-27 (1984)!.

Other less established control measures include the actual feeding oflive oocysts from well characterized wild type or attenuated strains ofseveral Eimeria species to chickens to establish immunity. Cocci-VacSterwin Labs! utilizes controlled numbers of specific species of chickenEimeria added to feed or water or administered individually See, e.g.,S. A. Edgar, Research in Coccidiosis, McDougald et al, eds., Universityof Georgia, p. 617 (1986)!.

Another approach to the development of a live vaccine includes theadministering of attenuated parasite strains. Selection for early oocystdevelopment or precociousness results in strains having abbreviatedasexual development, and reduced pathogenicity See, Shirley et al, AvianPath., 15:629 (1986); Shirley et al, Res. Vet. Sci., 44:25 (1988); andEuropean Patent No. 0256878-A2!. Serial passaging of Eimeria species inchicken embryos also results in strains of reduced pathogenicity Long,J. Comp. Path., 82:429 (1972); and Long, J. Comp. Path., 82:439 (1972)!.

Both attenuation practices have been used in combination with a "trickledose" method of administration to achieve effective immunity Johnson etal, "Research in Coccidiosis", McDougald et al, eds., University ofGeorgia, pp. 634-641 (1986)!. Although this method has been shown to beuseful for vaccination, it requires introduction and maintenance of liveparasites in a poultry operation that presents an inherent risk ofreversion to pathogenicity.

While active infection generates a protective immune response, effectiveimmunization with killed parasitic stages or structural antigens is lessclear. Early studies indicated that antigen extracts from dead parasiteswere not immunogenic Long et al, Exp. Parasitol., 16:1 (1965); Rose etal, "Vaccines Against Parasites", Taylor and Muller, eds., BlackwellScientific Publications, Oxford, pp. 57-74 (1980)).

In contrast, European Patent Application No. 0167443 describes anextract produced from sporulated E. tenella oocysts, which when injectedintramuscularly, protected chickens against homologous parasitechallenge. A similar extract produced from E. acervulina oocysts isdescribed in U.S. Pat. No. 4,724,145 which elicits a protective responseto challenge with that parasite, as well as E. maxima and E. tenella. Anexcysted extract of E. tenella sporozoites in an aqueous suspension forsubcutaneous administration is described in U.S. Pat. No. 4,808,404.

European Patent Application No. 0135712 describes solubilized E. tenellasporozoite antigens as effective immunogens; while European PatentApplication No. 0135073 refers to the use of antigens from solubilizedE. tenella merozoites as immunogens. European Patent Application No.0291173 describes sporulated E. tenella extracts for injection into theegg of the bird prior to hatching to induce immunity. U.S. Pat. No.4,863,731 describes the use of an aqueous concentrate of viablesporulated oocysts from at least one species of coccidia as a feedadditive.

In addition, antigen extracts from gametocytes of E. maxima are beingexamined for potential immunogenicity (See, e.g., European Patents No.0256514 and 0256536!. Although varied degrees of immunity have beendemonstrated with the above preparations, their preparation is highlylabor intensive and manufacturing practices are difficult on a largescale.

Recent and more practical approaches to vaccine development involve theproduction and characterization of genetically engineered antigensBinger et al, J. Cell Biochem., 10A:144 (1986); Brothers et al, Mol.Biochem. Parasitol., 28:235 (1988); Danforth et al, Avian Dis., 30:37(1985); Jenkins et al, Exp. Parasitol., 66:96 (1988); European PatentApplication No. 0164176; European Patent Application No. 0337589 andAustralian Patent Application No. 65867/86!. These procedures requirethe isolation of mRNA from sporozoites or merozoites, the production ofa CDNA library, screening of the cDNA library with an appropriateantibody, and the subsequent cloning into an expression vector. Theresultant cloned antigens can then be produced in large quantities inmicrobial fermenters.

Few immunogenicity studies have been reported to date, but suggestpartial protection is elicited by these antigens Danforth and Augustine,supra; Jenkins et al, supra!. Overall, these cloned structural proteinsinduce incomplete protection at best and their immunizing capabilitydepends in part on host genetics Clare, Infect. Immunol., 57:701(1989)!.

Finally, passive immunizations with monoclonal antibodies producedagainst E. tenella sporozoites U.S. Pat. No. 4,710,377! and activeimmunizations with anti-idiotypic monoclonal antibodies European PatentNo. 0241139! derived from E. tenella sporozoites are being investigated.

The advancement of knowledge on host/(protozoan) parasite interactionshas been hampered by the lack of adequate in vitro cell culture systemsin which to maintain parasites. Both mammalian and avian coccidia arevery difficult to grow in vitro, with the exception of Toxoplasmagondii, which grows well in a variety of primary cultures andestablished cell lines D. J. Doran, in "The Biology of the Coccidia",pp. 253-257 (1982)!.

In vitro propagation of Eimeria has to date been limited. The entireprepatent coccidia development from sporozoite to oocyst, has only beenobtained with E. tenella, and only in primary avian kidney cells Doranet al, J. Protozool., 20:658 (1973)!. However, the primary chickenkidney epithelial cell system is not compatible with manufacturingprotocols and has limitations for use as a research assay system.

Only one established cell line, Madin Darby Bovine Kidney (MDBK) hasbeen reported to support Eimerian growth in vitro, but the coccidiadevelops only through one generation of asexual development D. M.Schmatz, Adv. Cell Culture, 5:241 (1987)!.

Oocysts have been obtained from avian E. acervulina M. Nacri-Bontemps,Ann. Rech. Vet., 7:223 (1976)! and E. meleagrimitis Augustin et al, J.Protozool., 25:82 (1978)! as well as the bovine E. bovis Speer et al, Z.Parasitenkd, (1973)! when initial host-derived merozoites have been usedas the inoculum.

To date, no established cell line has been reported to support Eimeriangrowth beyond the first generation of asexual development. There remainsa need in the field of prophylactic and therapeutic treatment of variousavian pathogenic infections for an established cell line capable ofpropagating in vitro components of Eimeria species, to provide safe andeffective vaccines against these pathogens, including coccidia.

SUMMARY OF THE INVENTION

As one aspect, the present invention provides a novel continuous cellline, SB-CEV-1\P, which is capable of propagating avian coccidia. Alsodescribed are clones derived from this cell line.

Also part of this aspect of the invention are three additional celllines propagated from the aforementioned parental cell line. These celllines are referred to as SB-CEV-1\F7, SB-CEV-1\G7, and SB-CEV-1\A2.Clones, or sub-clones, derived from these cell lines are alsoencompassed by this invention.

As another aspect, the present invention provides the above cell linespersistently infected with an avian parasite, particularly a Coccidialparasite.

A further aspect of the present invention involves a novel method ofvaccine development in which coccidia antigens are produced at variousstages of asexual or sexual development by culturing one of the infectedcell lines and harvesting cell culture components for use in vaccinecompositions.

Yet another aspect of the present invention is a multicomponent vaccinecomprising selected pathogenic antigen compositions from various aviancoccidia pathogens produced through use of the cell lines describedherein.

Still a further aspect of the present invention is a vaccine forcoccidiosis capable of inducing host protection against infection inpoultry containing one or more of the vaccine compositions describedabove in association with suitable carriers and adjuvants.

Still a further aspect of this invention is a novel method forvaccinating poultry against infection by parasites causing coccidiosisinvolving administering to an animal an effective dose of theabove-described vaccine compositions.

Also involved in this invention is a method for producing a recombinantantigen by transfecting the cell line with a recombinant DNA moleculeencoding an exogenous protein under control of a suitable expressioncontrol sequence; and culturing the stably transfected cell line undersuitable culture conditions to produce the recombinant antigen.

Another aspect of this invention provides a method of drug screening foragents which destroy or inhibit the growth of the selected intracellularparasites comprising exposing the infected cell line of this inventionto a selected anti-infective agent, and examining any effects on thepathogen.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of preferred embodimentsof the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for prophylacticvaccination of Aves against infection by avian pathogens and parasites,particularly for the treatment and control of coccidia in poultry."Poultry" is defined herein to include birds of the order Galliformes,such as the ordinary domestic fowl or chicken (Gallus domesticus),turkeys (Meleagris), pheasants (Phasianus), partridges (Pedrix), grouse(Lagopus), guinea fowl (Numida) and peacocks (Pavo), and also birds ofthe order Anseriformes, such as ducks (Anas) and geese (Anser).

This invention provides a novel continuous cell line SB-CEV-1\P,described in detail in Example 1 below. This cell line has 42chromosomes per cell, and is reverse transcriptase negative. The cellline is characterized as containing only a low incidence ofnon-infectious viral particles (type A) associated with the endoplasmicreticulum. The cell line is also negative for endogenous mammalianpathogens, and has no indication of avian leukosis virus. Further thecell line has no contamination with mycoplasma, bacteria or fungi. Thusthe cell line is free of mammalian and avian viruses.

This cell line also has functional characteristics which associate itwith avian background. For example, this cell line replicates at 41° C.,characteristic of avian cells and it has unique nutritional requirementsfor maintenance in vitro. Further, the novel cell line of this inventionis the only existing continuous cell line capable of replicating theprepatent life cycle (i.e., that period between infection and thedetection of the parasite in the body) of the avian coccidia, Eimeria,at high levels.

The SB-CEV-1 cell line of this invention has been selected for theproduction of vaccine antigens, particularly avian coccidia. The cellline also provides substrates for use in the growth of geneticallyengineered vectors expressing recombinant DNA derived from foreigngenes.

Several cell populations have been cloned from this parent cell line.These clones have distinct characteristics for propagation andmaintenance of the avian parasite. Further these cloned cellpopulations, SB-CEV-1\F7, SB-CEV-1\G7, and SB-CEV-1\A2 have a highincidence of multinucleated giant cells. The appearance of distinctclones from the parent cell line is also indicative of a multicellularorigin, e.g., possible aberrant growth in the chicken viscera used asthe origin of the parent cell line. These cloned "progeny" cell lines,however, are also continuous cell lines capable of replicating theprepatent life cycle of the avian coccidia, Eimeria, at high levels.These cell lines are believed to share the same characteristics as theparental strain, and have also shown the ability to propagate Coccidia.This invention, therefore, also encompasses other cell lines which aresubcloned from, or otherwise derived from, SB-CEV-1\P or from thespecifically identified clones of that parent cell line. Such additionalprogeny clones are anticipated to share significant characteristics ofthe parent cell line. Thus the sub-clones may be substituted for theparent cell line wherever SB-CEV-1 or SB-CEV-1\P is specificallymentioned throughout this specification. Also, wherever in the followingdescription, a cell line is referred to in the singular, the term "thecell line" or "the SB-CEV-1 cell line", is meant to include SB-CEV-1\P,its subclones SB-CEV-1\F7, SB-CEV-1\G7, or SB-CEV-1\A2, or any othersubclones of any of these specifically identified cell lines.

The parent cell line and the subclones thereof of the present inventionmay be employed to support the in vitro development of avian Eimeriaspecies. While the disclosure below refers specifically to methods andvaccine compositions for E. tenella infections, it should be understoodthat other avian parasite pathogens, including viruses, as well as otheranimal species protozoans, may be produced using a cell line of thisinvention in analogous procedures. Thus the cell line is capable ofproviding an expression system for a variety of pathogenic antigens andother proteins for use in research, characterization and the productionof vaccine components. In addition, as the only existing continuous cellline that replicates the prepatent life cycle of avian Eimeria, the celloffers a unique substrate to study enzymatic and genetic characteristicsof a parasite permissive cell line.

The cell line of this invention also provides a means for production ofrecombinant avian vaccine components, such as subunit antigens derivedfrom reovirus, coronavirus, herpesvirus, para- and orthomycoviruses. Thecell line may be transfected by a recombinant DNA molecule or expressionvector encoding a selected pathogen protein or peptide under the controlof conventional regulatory control sequences, and cultured. Therecombinant protein may then be expressed by the cultured SB-CEV-1 cellline or its progeny.

The novel cell line also provides a substrate for replication of otherEimeria species. This continuous cell line of the invention may also beutilized to isolate and characterize independent stage specificcomponents of intracellular parasitic stages. Specifically, this cellline provides the only source of readily available parasite DNA, RNA,and protein from intracellular structures. Further, this cell line maybe used to grow other desired, selected pathogens.

The SB-CEV-1 cells of the present invention permit development of thechicken species E. tenella and E. necatrix, as well as development ofthe turkey coccidia, E. adenoides and E. meleagrimilis. It is expectedthat the cell line will also permit the development of other species,e.g., E. acervulina and E. maxima.

The presently preferred culture conditions for growth of the cell lineinclude culturing the cell line in Medium 199 Irvine Scientific! and 5%fetal bovine serum (FBS) (or equivalent such as Optimem and 1% FBS)under incubation conditions of 5% CO₂ and 40.5° C. The cells grow moreslowly at 37° C. rarely reaching confluency and require at least 10%serum. Other culturing conditions, including media formulations withregard to specific nutrients, oxygen tension and reduced serum, may beemployed for growth of these cells, and may be selected and optimized byone of skill in the art.

The novel cell line of this invention, SB-CEV-1\P, was deposited withthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md. on Jul. 3, 1990 under ATCC No. CRL10497. The development of thiscell line is described in detail in Example 1 below. The progeny cellline SB-CEV-1\F7 was similarly deposited on Jul. 3, 1990 under ATCC No.CRL10495. The progeny cell line SB-CEV-1\G7 was deposited on Jul. 3,1990 under ATCC No. CRL10496. These deposits comply with therequirements of the United States Patent and Trademark Office formicroorganism deposits made for patent purposes, and will be made tocomply with the requirements of the Budapest Treaty at the time offoreign filing.

The present invention further provides a variety of vaccine componentsand compositions prepared by the use of the cell line of this invention.A particularly desirable embodiment of this invention is a vaccinecomposition derived from Eimeria parasites. This vaccine composition maycontain whole cell extract (live or inactivated) from theabove-described cell lines infected with a selected pathogen, orsubfractions thereof. These vaccine compositions may also containmodified cellular or parasitic antigens, produced by modifying theculture conditions of the infected cell line.

In one embodiment, vaccine compositions for use in vaccines to aviancoccidiosis are developed by infecting a cell line of this inventionwith a selected parasite, preferably an Eimeria parasite, e.g., E.tenella. Infection of the cells is monitored by use of an in vitroenzyme-linked immunosorbent assay (ELISA) employing conventionallydeveloped monoclonal or polyclonal antibodies to various life cyclestages of the parasite. The infection may also be measured by aradiolabelled uracil uptake assay. Both the ELISA and uptake assays aredescribed in detail in Example 2 below.

Approximately 72 hours post-infection the cells and medium orextracellular secretions are harvested by collecting cells and/orculture fluids. As an optional step, if necessary, the culture fluidsmay be inactivated using conventional techniques, such as by sequentialfreeze/thaw cycles or by the addition of filtration, denaturing orcrosslinking agents such as β-propiolactone, formaldehyde orglutaraldehyde.

Various portions of this infected cell culture preparation may beemployed in vaccine compositions:

1) the whole preparation without subfractionation;

2) a modified preparation affected by changes in the culture media andconditions (e.g. the omission of serum during critical growth periods,pH or ion changes);

3) subfractionation to produce cell associated soluble components; and4) subfractionation and modified vaccine components.

One embodiment of a vaccine composition or component according to thisinvention is prepared by disrupting the infected cells of theabove-described culture by scraping. The resulting disrupted cellcomposition is used for a vaccine preparation without furtherdesiccation or hydration.

As another vaccine component of this invention the above-describedvaccine component may be modified by altering the serum concentration orcomponents and other nutritional additives, in the medium employed toculture the parasite-infected cell line of this invention. For example,early stages of the parasite may be arrested by culturing the infectedcell line in minimal essential medium, MEM. Alternatively, bysubstituting chemically defined media, the cell line will permit laterstages of parasite development to be employed in the vaccine. Additionalnutritional changes to the medium which may affect parasite developmentand modify the antigenic proteins produced in the cell line of thisinvention involve the addition or subtraction from the media of one ormore of biotin, choline chloride, insulin, or non-essential amino acids.

A modified vaccine component may also be produced in the infected cellline by the application of classical mutagenic techniques, e.g., theaddition of alkalating agents, chelating agents, dimerizing agents oroutside treatment of the cell line by ultraviolet light during theculture of the infected cell line. These agents may genetically modifythe cell and provide it with an altered capacity to produce an abnormalparasite. Alternatively, depending on the stage of development of theparasite within the cell when it is first contacted with the mutagenicagent, the parasite itself may be directly mutagenized for theproduction of a preferred vaccine component.

Still another embodiment of a vaccine composition for use inprophylactic treatment of Aves, particularly poultry, against Coccidiais prepared by the methods above using subfractions formed by thedisrupted cells and the medium from the cell culture. These fractionsare obtained by first separating the media from cell fractions, e.g., bycentrifugation, size, molecular weight, charge, or various conventionalbiochemical means. These fractions are then employed as vaccinecompositions which may be presented to birds. For example, one fractionof the above-described cell culture is obtained by centrifuging themedium containing the disrupted cells. The medium is removed, and theremaining material pelleted to obtain the cellular components. Thispellet is resuspended in fresh tissue culture media. In addition, thesupernatant fraction may also be utilized as a vaccine component.

One or more of the above described vaccine components can be admixed oradsorbed with a conventional adjuvant or administered without anadjuvant. The adjuvant is used as a non-specific irritant to attractleukocytes or enhance an immune response.. Such adjuvants include, amongothers, oil and water, aluminum hydroxide, muramyl dipeptide, killedBordetella and saponins, such as Quil A. Presently, the preferredadjuvant is Amphigen (Hydronics Inc.; U.S. Pat. No. 5,084,2693.

A preferred vaccine dosage is between approximately 0.05 μg-100 μg ofparasite protein. Other appropriate therapeutically effective doses canbe determined readily by those of skill in the art based on the aboveimmunogenic amounts, the condition being treated and the physiologicalcharacteristics of the animal. Accordingly, a pharmaceutical preparationprovides a unit dosage of between 0.1 to 2 mls of a sterile preparationof an immunogenic amount of the active vaccine components, or acombination thereof. In the presence of additional active agents, theseunit dosages can be readily adjusted by those of skill in the art.

A desirable dosage involves administration of 1 to 2 doses of desiredvaccine composition, where the antigenic content of each fraction isdesirably as stated above. The mode of administration of the vaccines ofthe invention may be any suitable route which delivers the vaccine tothe host. However, the vaccine is preferably administeredsubcutaneously. However, the vaccine may also be added to feed or waterfor ingestion in the form of a suspension. Other modes of administrationmay also be employed, where desired, such as intradermally,intravenously, or intramuscularly.

It will be understood, however, that the specific dose level, mode andtiming of administration for any particular animal will depend upon avariety of factors including the age, general health, and diet of theanimal; the species of the animal; synergistic effects with any otherdrugs being administered; and the degree of protection being sought. Ofcourse, the administration can be repeated at suitable intervals ifnecessary or desirable.

In preliminary tests of these vaccines, bird performance is enhanced.Preliminary results show that the above-described vaccine, which wasformed by simply decanting conditioned media or disrupting the SB-CEV-1cells, and harvesting by centrifugation the cell fluids, enhances birdperformance during challenge. Additionally the subfraction vaccinedescribed above also has shown efficacy in an in vivo assay. The in vivotests are conducted as follows. Chicks two weeks of age are immunizedsubcutaneously with 1 ml of culture media harvest. Two weeks later, thechicks are challenged with 10,000 oocysts of E. tenella. For the next 6days, the chickens are monitored for aspects such as weight gain andfeed efficiency. Intestinal lesions are thereafter screened and subjectsare scored on this basis. This assay is described in more detail inExample 4 below.

In addition to the use of the cell lines of this invention for thedevelopment of vaccines, these cell lines may also be used in methodsfor screening anti-parasitic agents in the development of newanti-coccidia drugs. For example, cultures of infected cells may beconventionally labeled, e.g., with a radioactive molecule. The selecteddrug for testing may then be incorporated into the cell cultures. Thecell culture may then be harvested at discrete intervals post infection,and label incorporation of the radioactive precursor may be determinedby harvesting and processing for scintillation counting. An example ofsuch drug screening employing alpha-amanitin as the test drug isdescribed in detail in Example 5 below. If a drug is effective at aparticular dose or time of administration, the incorporation of counts(parasite material) should be halted. If no inhibition of labelincorporation is observed, the drug is ineffective in controlling theparasite infection in vitro.

Other conventional drug screening modes known to those of skill in theart may also be employed using the cell lines of this invention.

The following examples illustratively describe the production of thenovel continuous cell lines of this invention. These examples are forillustration only and do not limit the scope of the present invention.

EXAMPLE 1 Isolation of Parental Cell Line, SB-CEV-1\P, and ClonesSB-CEV-1\F7 and SB-CEV-1\G7

SB-CEV-1 cells were isolated from an abnormal tissue mass (approximately1 cm×2 cm) associated with the visceral connective tissue of one 20 dayold SPAFAS COFAL-24! chicken embryo. The tissue was aseptically removedand rinsed in Hank's Basic Salt Solution (HBSS) containing 1% Fungi-BactSolution Irvine Scientific, Irvine, Calif.!. The tissue was minced withscissors, then enzymatically dissociated using 0.25% trypsin (1:250) inHBSS. The dissociated cell suspension was collected in a 50 mlcentrifuge tube containing 0.5 ml fetal bovine serum to inactive thetrypsin and centrifuged at 700 g for 10 minutes.

The cells were resuspended in 5 ml Weymouth's MAB87/3 media IrvineScientific! supplemented with 8 mg/l bovine insulin CollaborativeResearch, Inc., Bedford, Mass.!, 12 ml/l 200 mM L-glutamine and 1%Fungi-Bact Solution Irvine!. This 5 ml volume was pipetted into a 25 cm²Corning tissue culture flask and incubated at 40.5° C. in 5% CO₂. After24 hours of incubation, the media was changed. This primary culturecontained numerous explants with centers of epithelial-like cells andradiating fibroblasts.

After 72 hours, the near-confluent culture was washed once with Ca⁺⁺/Mg⁺⁺ -free phosphate buffered saline (PBS) then treated with 0.02%ethylene diamine tetraacetic acid (EDTA) in HBSS to dissociate thecells. The resulting cell suspension was decanted, the cells werecollected by centrifugation and resuspended in the MAB87/3 mediaformulation used previously. This culture was then split 1:10, creatingpassage-1 (P1) by plating cells in seven 25 cm² culture flasks and two60 mm² petri dishes. Cultures were incubated as before.

Media was changed on actively growing cultures at 72 hours omittingfetal bovine serum (FBS). The media in one flask was replaced withMedium 199 Irvine Scientific! supplemented with 10% FBS. After anadditional 48 hour incubation, the culture containing Medium 199 showedactively growing cells, while the MAB87/3 cultures were static.Returning serum to these cultures up to 10% did not promote cell growthto the level observed using Medium 199. Therefore, all subculturing wasdone, hereafter, in Medium 199 plus 10% FBS.

Further subculturing was done (Passage-2 to Passage-11) when flasksreached confluency. With increasing passage number, cells grew moreslowly, became fibroblastoid and highly vacuolated and released debrisinto the media. Additional media formulations (EMEM+10% FBS; RPMI1640+10% FBS; DMEM/Ham's F-12+5% FBS) were tested on these cells toforestall or counteract this pending senescence. However, cells showedthe least deterioration in Medium 199. Cells from several passages (P4,P5, P6, P7, P9, P10) were frozen down in liquid nitrogen. These cellsappeared to reach crisis at P11-13 and died.

One 75 cm² flask of P11 cells containing a very few isolated foci ofcells was repeatedly fed with Medium 199 and 10% FBS for 58 days afterits last subculturing. At this point, fibroblast-like cells began togrow outward from these foci. After an additional 15 days, cells in thisT-75 flask reached confluency and were split 1:2 creating P12.

Subculturing has continued to the present in Medium 199 GibcoLaboratories, Grand Island, N.Y.!, 3.43 ml/l of 200 mM L-glutamine and1% Antibiotic-Antimycotic Gibco Laboratories! using the passagecriterion of splitting 1:20 every 7 days. The cells have lost allepithelial characteristics and are distinctly fibroblast-like inmorphology. Other media formulations used successfully since crisisinclude Weymouth's MAB87/3 and 5% FBS, Dulbecco's MEM GibcoLaboratories! and 5% FBS and MEM with Earle's salts Gibco Laboratories!and 5% FBS. The FBS requirement has been reduced to 5% for SB-CEV-1cells of passage 24 which were subjected to subcloning by dilution usingsingle-cell isolation in 96 well microculture plates. This techniqueproduced 25 clones from the parental cell line SB-CEV-1\P inunconditioned Medium 199 with 5% FBS. Of these clones, two, designatedSB-CEV-1\F7 and SB-CEV-1\G7, showed an exceptional capacity to supportasexual development of E. tenella.

These two clones along with the parental line, SB-CEV-1\P have beendeposited with the ATCC as identified above. Passage No. 10 wasdeposited for both clones and passage No. 20 for the parental line.Freezing has not had any deleterious effect on cell performance asseveral frozen samples have been restored successfully. Standardbiologic quality control was satisfactory from P33 of the parent line.In addition, the parent line shows a karyotype of approximately 42chromosomes, is reverse transcriptase negative, does not express avianretroviruses (e.g. avian leukosis), does not express other endogenouspathogens (mammalian or avian), is tumorigenic in nu/nu mice, and showsno bacterial, fungal or mycoplasma contamination. A low incidence ofA-type viral particles associated with the endoplasmic reticulum wasresolved by transmission electron microscopy. The parent line as well asboth clones showed isoenzyme focusing profiles similar to BHK-21 cellsNational Veterinary Services Laboratory! and dissimilar to SL29 cells (atransformed chicken fibroblast line) ATCC No. CRL1590! for the enzymeslactose dehydrogenase, malate dehydrogenase, nucleoside phosphorylase,peptidase A and phosphoglucomutase. In contrast, both clones and theparent SB-CEV-1 line are morphologically distinct from BHK-21 andACC-111 cells. In addition, the SB-CEV-1 cell lines show a highincidence of multinucleated giant cells. Most importantly, the SB-CEV-1cells produce high levels of parasite material from different stages ofthe Eimeria prepatent life cycle.

EXAMPLE 2 Assays to Monitor Parasite Development in the Cell Line

A. The direct sporozoite-based enzyme linked immunosorbent assay fordetection of coccidia proteins (SPZELISA) involves the adherence ofantigen (e.g., supernatants from uninfected or infected F7 cells ordisrupted sporozoites (SPZ) or merozoites) in twofold serial dilutionsto the well of a 96-well tray. Antibodies which recognize SPZ antigensbind to the antigen in a dose related response. After the primaryantibody is bound, a second antibody produced in goats against rabbitIgG which is also biotinylated is added. Again, this anti-rabbitantibody will bind to the rabbit #15, 16 anti-SPZ antibody previouslybound to antigen in the wells. The anti-SP2 antibodies were produced asfollows. Purified sporozoites (described below) for E. tenella weresuspended in serum free media at a concentration of 2-5×10⁶ sporozoiteper ml. An equal volume of Freunds Complete Adjuvant was added mixed and0.5 ml was inoculated subcutaneously at 2-4 sites on the back of 6 kgNew Zealand white rabbits (mixed sexes). Booster inoculations were givenin a similar manner using Incomplete Freunds Adjuvant at 2 to 4 weekintervals (3 inoculations minimum). Blood was collected in serumvacutainers (Becton-Dickenson) two weeks after third inoculation. Theserum was allowed to clot at room temperature for 1 hour and then wascentrifuged at 2000 rpm for 10 minutes to pellet the clot. The serum wasremoved, aliquoted into 1.5 ml per tube and stored at -20° C. After thesecondary antibody is bound, an enzyme-labelled streptavidin which bindsto biotin is added. Substrate is incubated and the enzyme linked tostreptavidin bound to the well will convert the substrate to a visibleform. The amount of color measured is proportional to the amount ofantigen cross-reactive to SPZ proteins in the test supernatant. Includedon the plate are samples containing antigens from uninfectedsupernatants as a negative control as well as sonically disrupted SPZ.This sporozoite material serves as a positive control and is used togenerate a standard curve against which parasite antigens in infectedsupernatants are measured. In this manner, parasite-specific material ininfected cell supernates can be quantitatively assessed and compared.

Approximately fifty 3-4 week old birds are each infected orally with100,000 E. tenella oocysts. Cecal pouches are harvested at approximately7.5 days and lumen contents subjected to pepsin digestion. Oocysts thenundergo sporulation in 2.5% potassium dichromate for 3-4 days and aresterilized by chlorine bleach. The sterilized oocysts are stored at 4°C. in Medium 199+2× antibiotic. Generally, fifty birds yieldapproximately 5×10⁹ oocysts. This protocol is repeated every 3-4 weeksto maintain virulence.

Cell culture antigen for the assay is produced according to thefollowing modified excystation procedure. The sporocysts are purified bybreaking 10 ml oocysts (at 3×10⁷ /ml) with 5 ml 0.5 μm glass beads in asmall chamber bead beater and separating the debris using 0.75M sucrosein PBS followed by centrifugation using 50% isotonic Percoll. Using asolution consisting of 4% (w/v) tauradeoxycholic acid, 0.25% (w/v)trypsin, HBSS, and adjusting to pH 8.0 with bicarbonate, SPZ excystationis performed by incubating this mixture with the purified sporocysts at40.5° C., 60-90 minutes with vortexing at approximately 15 minuteintervals. The SPZ are then collected using 60% isotonic Percoll, thepellet is resuspended in serum-free media and counted. Generally, 3×10⁸oocysts yields approximately 7.2×10⁸ SPZ (30%). The SPZ are sonicatedand stored at -20° C. as a standard source of antigen for the assay.

The antigen seeding is performed as follows. 200 μl of antigen preparedin 10 mM borate buffer, pH 9.0, is added to the top wells of a 96 wellNunc Immuno plate. All remaining wells contain 100 μl of the boratebuffer alone. Serial 2-fold dilutions are made in rows B-G. Row Hcontains only buffer and is used as a negative control. The wells arecovered with parafilm and incubated overnight at 4° C. The SPZ controlis loaded at 10 ng in the top row of wells. Antigen without 1% FBS isloaded at 100 ng and antigen+1% FBS Gibco! is loaded at 1000 ng.Supernatant antigens from uninfected F7 cells are included as a negativecontrol in each assay. Supernatants harvested at 72 hours from adesignated infected passage of F7 cells are also included on each plateas an internal standard. The 72 hours supernatant is first quantitatedby the SPZELISA and then its relative value as compared to the SPZcontrol is used to monitor and adjust for assay-to-assay variability.

Next, supernatants are washed 3× with PBS+0.05% Tween-20 (PBS-T) andblocked by adding 200 μl of 5% skim milk (Difco) in PBS-T to each well.Wells are incubated 1 hour at 37° C. covered with plastic wrap, and arewashed again 3× with PBS-T. The primary antibody is then added. 100 μlof rabbit anti-SPZ antibodies #15, 16 diluted to 1:20,000 in 0.5% BSA inPBS-T is added per well and the plates are incubated 1 hour at 37° C.covered with plastic wrap. The plates are washed again 3× with PBS-T.Following this, the conjugated antibody, 100 μl of 1:2000 dilution ofbiotin-labeled goat anti-rabbit IgG (KP) in 2% skim milk in PBS-T, isadded per well. The plates are again incubated 1 hour at 37° C. and thenwashed 3× with PBS-T. Following this, 100 μl of 1:1500 dilution ofperoxidase-labeled streptavidin Kirkegaard Perry! in 2% skim milk inPBS-T is added per well. The plates are then incubated 1 hour at 37° C.in the dark and washed 3× with PBS-T. TMB-Peroxidase Kirkegaard Perry!is mixed in a 1:1 ratio with H₂ O₂ and 100 μl of substrate per well isadded. The plates are then incubated 15 to 30 minutes at 37° C. in thedark. At the end of the incubation time, 100 μl of 1M HCl per well isadded to stop the reaction. Readings are taken at 450 nm on the Vmax.

B. Another assay employed to monitor parasite development in the cellstakes advantage of the parasite's, but not the host's, ability toincorporate radiolabelled uracil into its RNA D. M. Schmatz et al, J.Protozool., 33:109-114 (1986)!. Briefly, cultures of cells in microtiterplates are seeded at 1×10⁵ cells/ml, 0.1 ml/well, 24 hours prior toinfection with E. tenella at 1×10⁵ sporozoites per well. The sporozoitesare incubated with the cells for 4 hours at 40.5° C. and then removed bywashing with serum-free medium. The cells are then overlaid with mediumand serum and incubated for 24 hours. At 24 hours post-infection, thecells are washed and then refed with medium containing 3H!-uracil. Labelis incorporated over a 24 hour period and then the cells are collectedonto filters using a cell harvester (Cambridge Technology, Inc.).Radioactivity on the filters is determined in a Beckman LS 3801 liquidscintillation counter after the addition of aqueous scintillationcocktail (Beckman Ready Safe). Background counts and radiolabelincorporated into uninfected cells are also measured.

EXAMPLE 3 Avian Vaccines

A. One vaccine formulation is prepared from a SB-CEV-1/P host cell cloneseeded at a rate of 1.0×10⁵ cells/ml in a T-150 flask containing 30 mlof either Medium 199 containing 5% FBS Irvine Scientific! or OptiMEMcontaining 1% FBS. E. tenella sporozoites, excysted by conventionaltechniques known to those skilled in the art, are used as inoculum 24hours later at a rate of 1×10⁶ /ml. The sporozoites are left to invadefor 2 hours, after which non-invaded sporozoites are removed by gentlewashing. Fresh media is added to each flask. At 24 hour intervals postinfection, the culture media is collected, centrifuged at 3000 × g for30 min, and adjuvanted with 5% Amphigen. This formulation (designated 24hour supernate, 48 hour supernate and 72 hour supernate, etc.) is storedat 4° C. until use.

B. An alternative formulation utilizes the remaining cells from theabove-described vaccine. A volume of 30 ml fresh media, Medium 199 orOptiMEM, is added to the T-150 flask, and the cells are scraped intosuspension. This suspension is collected, subjected to a freeze/thawcycle, and adjuvanted with 5% Amphigen. This formulation is stored at 4°C. until use.

C. A still alternative formulation utilizes the entire infected culturefrom the above-described vaccine, unfractionated. Upon harvest, theinfected cells are scraped into suspension. This suspension iscollected, subjected to a freeze/thaw cycle, and adjuvanted with 5%Amphigen. This formulation is stored at 4° C.

EXAMPLE 4 Imunogenicity Data

A. Broiler Immunogenicity Study #1

A study was conducted to screen E. tenella cell culture-derived antigensfor immunogenicity in commercial broilers comparing Amphigen andFreund's Complete Adjuvant (FCA) as adjuvants for the primaryimmunization.

Three hundred 4 day old straight run commercial broiler chicks weredivided among twenty groups (15 birds per group and wing-banded) asfollows. PSP refers to parasite-specific protein which is quantitatedusing the direct SPZELISA described in Example 2 above.

    ______________________________________                                              TREATMENT-               TREATMENT-                                     GROUP AMPHIGEN   PSP     GROUP FCA      PSP                                   ______________________________________                                        1A    Unchallenged                                                                             0 μg/ml                                                                            1B    Unchallenged                                                                           0 μg g/ml                                control                  control                                        2A    Challenged 0       2B    Challenged                                                                             0                                           control                  control                                        3A    24 h antigen                                                                             0.70    3B    24 h antigen                                                                           0.35                                  4A    48 h antigen                                                                             1.40    4B    48 h antigen                                                                           0.70                                  5A    72 h antigen                                                                             1.40    5B    72 h antigen                                                                           0.70                                  6A    24/48 h (1:1)                                                                            1.10    6B    24/48 h (1:1)                                                                          0.60                                  7A    24/48/72 h 1.20    7B    24/48/72 h(1:1)                                                                        0.60                                        (1:1:1)                                                                 8A    24 h primary/                                                                            0.7/1.4 8B    24 h primary/                                                                          0.35/0.7                                    48 h boost               48 h boost                                     9A    5 × 500 Trickle                                                                            9B    5 × 500 Trickle                          ______________________________________                                    

Chicks in Groups 1-8 were immunized subcutaneously (sc) at 4 days of ageas designated, and boosted orally with the same amount of antigen in 5%Amphigen at 7 days of age. Both control groups received 1 mlinoculations of tissue culture medium (Gibco Medium 199+1% FBS)adjuvanted to 5% Amphigen or 1:1 with FCA SIGMA!. Antigen for Groups 3-8was prepared from host cell clones F7(P24-31) for the 24 hour antigenand F7(P24-29) for both 48 hours and 72 hours antigens. Antigens werestored at -20° C. until use or subjected to one freeze-thaw cycle.

E. tenella oocysts for groups 9A and 9B were administered orally, 500oocysts per day for 5 consecutive days (Lilly Strain #65 strain, Lilly,Colo.) gift from University of New Hampshire (UNH)!. In addition, group9B birds received a s.c. injection of 50% FCA at 4 days of age.

Groups 2-9 were challenged with 35,000 L.S. #65 E. tenella oocysts(number determined by titration) at 21 days of age. At this time, bodyweights of all chicks were measured, and feed consumption monitoredduring the prepatent period. Six days post challenge, body weights, feedconsumption and cecal lesion scores were measured.

The clinical data from Trial #1 has been summarized in Tables 1 and 2.All least squares statistical comparisons for weight gain were madebetween vaccinates the unimmunized/challenge (UI/C) control group. Maineffects tested included Treatment, Pens within Treatments, Sex, and aSex by Treatment Interaction. Both Amphigen and FCA data sets weretested separately. For both data sets a significant interaction withtreatment permitted analysis across sexes. No sex effect was measuredfor lesion score. Feed conversion was tested for only treatment effects.

In the following table, UI/UC means UnImmunized/UnChallenged; and Agrepresents antigen.

                  TABLE 1                                                         ______________________________________                                        Clinical results of Trial #1 - Amphigen                                                       WEIGHT    FEED       LESION                                   TREATMENT                                                                              N      GAIN      CONVERSION SCORE                                    ______________________________________                                        UI/UC    13     287       1.8        0                                        UI/C     13     223       2.1        2.5                                      24 hour Ag                                                                             14     255       1.9        2.9                                      48 hour Ag                                                                             14     223       2.1        2.9                                      72 hour Ag                                                                             15      264#      1.5*      2.8                                      24/48 hour Ag                                                                          14      261#     2.0        2.8                                      24/48/72 hour                                                                          14      262#     2.0        2.6                                      24->48 hour                                                                            15      273*     1.9        2.6                                      Trick1e   6     257       1.8        2.8                                      ______________________________________                                         *p < 0.05                                                                     #p < 0.1                                                                 

                  TABLE 2                                                         ______________________________________                                        Clinical results of Trial #1 FCA                                                              WEIGHT    FEED       LESION                                   TREATMENT                                                                              N      GAIN      CONVERSION SCORE                                    ______________________________________                                        UI/UC    15     286       1.8        0                                        UI/C     15     243       2.0        2.7                                      24 hour Ag                                                                             15     260       1.9        3.0                                      48 hour Ag                                                                             14     233       2.1        2.6                                      72 hour Ag                                                                             15     231       2.6        2.8                                      24/48 hour Ag                                                                          14     253       1.9        2.9                                      24/48/72 hour                                                                          15      196*     2.3        2.9                                      24->48 hour                                                                            15     215       2.2        2.5                                      Trickle  15     266       1.9         1.5*                                    ______________________________________                                         *p < 0.05                                                                

Amphigen adjuvanted cell culture antigens administered sc at 4 days ofage and orally at 7 days of age elicited significant (p<0.05) or nearsignificant (p<0.1) weight gain protection to a 35,000 E. tenella oocystchallenge in battery cages. The regimen of 24 hours antigen s.c.followed by 48 hours antigen orally elicited significant weight gainperformance, while gains sustained by the 72 hours antigen andcombinations 24/48 hours and 24/48/72 hours approached significance.None of the Amphigen adjuvanted treatments affected a reduction inlesion scores. Only the 72 hours antigen vaccinated group showed asignificant enhancement of the feed conversion ratio. The trickle oocystimmunized groups did not test significant for protection.

Cell culture adjuvanted with FCA elicited no significant protection tochallenge, measured by weight gain or feed conversion. In fact the groupimmunized with the 24/48/72 hours antigen combination had significantlylower weight gains than the challenge control group. Only the trickleoocyst group had a significant reduction in lesion scores.

The following conclusions can be drawn from this data. The regimen of 24hours s.c./48 hours oral vaccine elicited significant weight gainprotection to challenge when adjuvanted with Amphigen. The 72 hoursantigen and combinations of 24/48 hours and 24/48/72 hours antigens, alladjuvanted with Amphigen, also showed indications of protection in termsof weight gain. These findings suggest that each antigen preparationcontains either a different composite of antigens or a different ratioof similar antigens.

Weight gain protection was measured in the absence of any reduction inlesion scores, indicating that these parameters are affected bydifferent mechanisms. Weight gain and feed conversion performance may besustained even in the presence of cecal lesions.

While the 48 hours antigen alone was ineffective, this antigen incombination with the 24 hours and/or 72 hours antigens or administeredorally at 7 days may be critical to establishing immunity to challenge.It is assumed that the 72 hours antigen harvest contains a composite ofantigens representative of all three time points.

FCA was not successful in potentiating immunogenicity of cell cultureantigens. FCA alone may be eliciting a non-specific response tochallenge as indicated by the higher challenge control weight gains inthe FCA data set.

The importance of the oral dose, its time of administration, andsubsequent impact on performance in a floor pen design including agrow-out following challenge is evaluated in the following studies (partB).

B. Broiler Immunogenicity Study #2

The purpose of this study was to screen several E. tenella cellculture-derived antigens for immunogenicity in commercial broilers usingfloor pens, and including a 40 day grow-out.

Two hundred fifty 4 day old male commercial broiler chicks were dividedamong ten groups (25 birds per group and wing-banded) as follows. In thetable, UI/UC/Med means UnImmunized/UnChallenged/Medicated; UI/UC/Unmedmeans UnImmunized/UnChallenged/UnMedicated; and UI/C/Unmed meansUnImmunized/Challenged/UnMedicated.

    ______________________________________                                        GROUP       TREATMENT       PSP per dose                                      ______________________________________                                         1          UI/UC/Med       0 μg/ml                                         2          UI/UC/UnMed     O                                                  3          UI/C/UnMed      0                                                  4          24 hours, 4 d   2 μg/ml                                         5          24 hours, 4 d/7 d                                                                             2 μg/ml                                         6          72 hours, 4 d   2 μg/ml                                         7          72 hours, 4 d/7 d                                                                             2 μg/ml                                         8          24/48 hours, 4 d                                                                              2 μg/ml                                         9          24/48 hours, 4 d/7 d                                                                          2 μg/ml                                        10          24 hours, 4 d   2 μg/ml                                                    48 hours, 7 d   1.6 μg/ml                                      ______________________________________                                    

All chicks were held on wire until 4 days of age. At that time, chicksin Groups 1-10 were immunized s.c. and placed into clean litter floorpens as designated. Chicks in Groups 5,7,9 and 10 were boosted orally asdesignated at 7 days of age. Control groups received 1 ml inoculationsof tissue culture medium (Gibco Medium 199+1% FBS) adjuvanted to 5%Amphigen. In addition, Group 1 was given feed medicated with stenerol,at 3 ppm, throughout the study. Antigen for Groups 4-10 was preparedfrom host cell clones F7(P24-24) and adjuvanted with 5% Amphigen.

All chicks were fed a starter ration through 27 days of age, andswitched to a grower ration for the day 27-40 grow-out. Feed and waterwere provided ad libitum.

All birds in groups 3-10 were challenged with 35,000 (dose determined bytitration) L.S. #65 E. tenella oocysts at 21 days of age. At this time,body weights of all birds were measured, and feed consumption monitoredduring the prepatent period.

Six days post challenge, body weights and feed consumption weremeasured. In addition, five birds from each pen were selected randomlyfor cecal lesion scoring. All remaining birds were switched to a growerration and continued until 40 days of age. During this time both weightgain and feed consumption were monitored. At day 40, all birds weresacrificed for cecal lesion scoring.

The clinical data from Trial #2 has been summarized in Table 3. Allleast squares statistical comparisons for weight gain were made betweenthe unimmunized/challenge (UI/C) control group and each individualtreatment group (not with the medicated control group). No statisticswere performed on lesion score or feed date (one observation/group).

                  TABLE 3                                                         ______________________________________                                        Clinical results of Trial #2                                                            (21 d-                  (27 d-                                                27 d)                   40 d)                                       TREATMENT GAIN     FEED    LESIONS                                                                              GAIN   FEED                                 ______________________________________                                        UI/UC/MED 378      1.84    0/0    848    2.20                                 UI/UC/UNMED                                                                             324      1.97      0/2.4                                                                              764    2.34                                 UI/C/UNMED                                                                              334      1.91    2.4/0.8                                                                              719    2.48                                 24 h-4 d  307      1.98    2.0/1.6                                                                              734    2.49                                 24 h-4 d/7 d                                                                            336      1.90    2.8/2.0                                                                               791*  2.26                                 24 h-48 h-4 d                                                                           301      2.01    3.0/2.2                                                                              774    2.32                                 24/48 h-4 d/7 d                                                                         283      2.26    1.0/0  767    2.37                                 24 h->48 h                                                                              250      2.52    1.2/0.4                                                                               787#  2.17                                 72 h-4 d  282      2.10    2.2/0.4                                                                               781#  2.16                                 72 h-4 d/7 d                                                                            342      1.82    2.2/1.4                                                                               894** 1.98                                 ______________________________________                                         #p < 0.05                                                                     *p < 0.01                                                                     **p < 0.001                                                              

Prior to this study, E. tenella had not been used experimentally in thisset of ten floor pens, and no cecal lesions were detected in theUI/UC/Unmed group (there was a possibility that E. tenella could havebeen cycling prior to challenge in the other pens). However, E.acervulina was used previously in the same set of floor pens, and upperintestinal lesions characteristic of this species were detected in theUI/UC/Unmed group.

Although not statistically significant at 6 days post challenge, onlythe group receiving the 72 hours antigen 4 day s.c./7 day oral showedweight gain higher than challenge controls and feed conversions lowerthan the medicated control group. Following the 40 day grow-out, thissame 72 hours antigen vaccinated group showed highly significant(p≦0.001) protection over challenge controls in terms of weight gain,and a lower feed conversion ratio. In addition, the 24 hours antigenadministered s.c. at 4 days followed by the 48 hours antigen orally at 7days all elicited significant protection in terms of weight gain overchallenge controls and comparable or better feed conversions than themedicated controls following the 40 day grow-out. The 24 hours 4 day/48hours 7 day antigen regimen (group 10) as well as the 24/48 hours 4 days.c./7 day oral treatment elicited lower intestinal lesion scores 6 dayspost challenge. No intestinal lesions were detected following thegrow-out, although a general thickening of the mucosa was observed inthe challenge control group. It is reasonable to assume that protectionagainst E. tenella, measured during the grow-out, was elicited in thepresence of cycling E. acervulina.

The following conclusions can be drawn from the data. Performanceprotection (weight gains) may be difficult to measure in floor pens 6days following oocyst challenge at 21 days of age. A grow-out to atleast 40 days may be required to demonstrate significant vaccineefficacy in floor pens.

The 72 hour antigen given once s.c. at 4 day or twice 4 day s.c./7 dayoral elicited significant protection over challenge controls. The 72hours antigen given twice sustained performance comparable to thatmeasured in the medicated control group. This protection wasdemonstrated in the presence of a 35,000 E. tenella challenge and E.acervulina cycling in the litter. This is the first demonstration of aninactivated coccidiosis vaccine efficacy in a floor pen system.

The 24/48 hours antigen administered 4 day s.c./7 day oral and theregimen of 24 hours antigen given 4 day s.c. and the 48 hours antigengiven 7 day oral elicited the greatest reduction in intestinal lesionscores for both E. acervulina and E. tenella. This two dose regimen 4day S.C. followed by 7 day oral appears to be better than a single s.c.immunization at 4 day.

EXAMPLE 5 In Vitro Drug Screening

Microcultures of infected cells (in presence of ³ H-uracil) wereestablished at time T=0, using log₂ dilutions of alpha-amanitinbeginning with 50 μg/ml. Cultures were then harvested at 1 hour, 6hours, 12 hours, 24 hours and 48 hours post infection, and labelincorporation of radioactive precursor was determined by harvesting andprocessing for scintillation counting. When alpha-amanitin was presentduring the first 24 hours of parasitism, incorporation of counts(parasite material) was halted. However, if the alpha-amanitin was addedafter 24 hours, no inhibition of label incorporation was observed.

EXAMPLE 6 Immune Measurements

One-day old inbred chickens (B¹⁹ B¹⁹ and B³⁰ B³⁰ MHC haplotype) NewHampshire Poultry Research Center!, originally derived from the UCD.003line, were used. Chicks were fed a nonmedicated starter/grower diet andwater ad libitum. Birds were used between 1 and 43 days of age.

To simulate natural immunity, one-day old chicks were immunized withlive E. tenella (Lilly Strain #65) oocysts for five consecutive days(500 oocysts/day) or artificially immunized at various doses withvaccine antigens (adjuvanted to 5% Amphigen). Typically, 1- or 4-day oldbirds were immunized subcutaneously (s.c.) in a 1.0 ml volume at thebase of the neck and then boosted with vaccine adjuvanted antigens at 4or 7 days of age by oral gavage in a 1.0 ml volume. Sham immunized(media plus 5% Amphigen) chickens were used as controls. In someexperiments, chickens were challenged at 10 days of age by oralinoculation with 35,000 E. tenella oocysts.

A. Vaccine and Parasite antigens

Media from E. tenella infected F7 cells, collected at 24, 48 and 72hours post-infection was used as the source of antigen for immunizationsand for in vitro assays. For immunizations, media collected frominfected F7 cells contained 1% FBS and for in vitro assays infectedmedia collected was serum-free (0.1% FBS). Antigen-containing media wasclarified by centrifugation (800× g, 30 minutes, 4° C.), aliquoted andstored at -20° C. until use. All cell-free supernatants (SN's) werequantitated for parasite-specific protein (PSP) using the directSPZELISA. Fractionated samples were pooled according to PSP and Westernreactivity. Sporozoite (SPZ) and merozoite (mrz) antigen were preparedby sonication on ice in serum-free 199 media followed by centrifugation(800× g, 10 min, 4° C.). Protein concentrations were determined by themethod of Bradford, Anal. Biochem., 72:248-252 (1976). Sonicatedparasite suspensions were adjusted to a final concentration of 10 μg/mLin serum-free Medium 199, aliquoted, and stored at -20° C. until use.

B. Cell Isolation

Peripheral blood lymphocytes (PBL) and spleen cells were obtained fromnaturally or artificially (vaccine)-immunized or immunized/challengedbirds at various time points post-immunization. PBL obtained by cardiacpuncture were isolated by Histopaque 1077 (400× g, 15 minutes, roomtemperature) centrifugation of heparinized blood samples. In someassays, red blood cells from gradients were also saved and used asco-stimulants for in vitro proliferation assays. Single cell spleensuspensions were obtained by disruption of minced tissues by syringecannulation, followed by slow speed centrifugation (50× g, 10 minutes,room temperature) and subsequent centrifugation over Histopaque 1077gradients. Viable cell counts were performed using trypan blue and ahemacytometer.

C. Production of Antigen and Mitogen-Stimulated Cell Supernatants

Undiluted serum-free antigens, various concentrations of Conconavalin A(Con A) or alipopolysaccharide (LPS), or serum-free Medium 199 (1.0ml/well) were cultured with lymphocytes at 40° C., 5% CO₂. After 24, 48and 72 hours supernatants were removed from wells and clarified bycentrifugation (800× g, 15 minutes, room temperature). α-methylmannoside (α-MM) was added to Con A-containing supernatant to a finalconcentration of 50 mM. Supernatant samples were aliquoted into 1.5 mltubes and stored at -80° C. until use. Media produced from 0-48 hour(24/48) or 48-72 hour (72) post-infection was collected, fractionated byS-Sepharose using a linear NaCl gradient, and pooled based on PSP andWestern reactivity. Fraction I represents a pool of 8 fractions takenfrom the first part of the total fractions collected. Fraction IIrepresents a pool of fractions numbers 9-14 and Fraction III a pool from15-18.

D. Cell Proliferation Assays

Ten μg total protein from each fraction was assayed for reactivity inthe cell proliferation assay described below.

Cells were adjusted to 10⁷ cells/ml in complete serum-free Leibovitz'sModified Hahn's media (cLMH) which contains equal parts McCoy's 5A andLeibovitz's media, 5×10⁻⁵ 2-mercaptoethanol, 5 ug/ml insulin, 2 mML-glutamine, 100 U/ml penicillin/streptomycin, 0.25 ug/ml amphotericinB, 2% tryptose phosphate, and 1 mM sodium pyruvate. Red blood cells (10⁷/ml) were added (0.05 ml/well) to all wells of round-bottomed microliterplates. Undiluted serum-free antigens, Con A, or serum-free Medium 199(0.1 ml/well) were thawed in a 37° C. water bath and added inquadruplicate, followed by PBL or spleen cells (0.05 ml/well). Formitogen and antigen proliferation assays, cultures were incubated at 40°C., 5% CO₂ for 72 or 96 hours respectively, and then pulsed with 1μCi/well³ H!-thymidine (specific activity, 5.0 μCi/mmol) during thefinal 18 hours of culture. Cells were harvested onto glass fiber matsusing a MACHIII harvester and radioactivity determined in a PackardMatrix 96 Direct Beta Counter. High and low cpm for each sample werediscarded. Except where specified, results were expressed by astimulation index (S.I.) according to the formula: ##EQU1##

Splenic lymphocytes from naturally immune birds were found toproliferate in response to E. tenella-infected-biochemically separatedfraction II from a S-Sepharose column. Splenic lymphocytes obtained from25-day old naturally immune birds showed higher S.I. values to fractionII compared to fractions I and III (Table 4).

                  TABLE 4                                                         ______________________________________                                               Fraction                                                                              S.I. Value                                                     ______________________________________                                               24/48  I    1.1                                                               72     I    0.6                                                               24/48  II   2.8                                                               72     II   2.5                                                               24/48 III   2.0                                                               72    III   0.6                                                        ______________________________________                                    

E. T-cell Westerns

Pooled fraction 72-II was separated by one-dimensional SDS-PAGE andtransferred to nitrocellulose, solubilized and assayed for reactivity inthe T-cell Western proliferation assay as described below.

One-dimensional immunoblotting studies were conducted by using amodification of the method of Lamb and Young, Immunol., 60:1 (1987).Briefly, pooled fractions from S-Sepharose were separated by sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) underreducing conditions on 10 or 12.5% acrylamide mini-gels. The separatedproteins were transferred to nitrocellulose (pore size, 0.2 μM) and thenitrocellulose cut into 12 equal sections corresponding to differentmolecular weight ranges. Nitrocellulose pieces were solubilizedaccording to the method of Abou-Zeid, J. Imm. Methods., 98:5 (1987)using DMSO/carbonate-bicarbonate precipitation followed byfreeze-thawing in deionized H₂ O. Solubilized, microparticulate sampleswere washed 3× in serum-free cLMH and resuspended in a final volume of1.0 ml serum-free cLMH. Samples were stored at -20° C. until use. Forassays, samples were thawed at room temperature, diluted 1:5 inserum-free cLMH, and added in quadruplicate (0.1 ml/well) toround-bottomed microliter plates containing 5×10⁵ red blood cells (0.05ml/well). PBL or spleen cells (10⁷ cells/ml, 0. 1 ml/well) were thenadded to plates and cultures incubated at 40° C., 5% CO₂. Cultures werepulsed with 1 μCi/well³ H!-thymidine during the final 18 hours ofculture, harvested and counted as previously described. Results areexpressed as S.I. in Table 5 according to the above formula.

PBL from naturally immune birds also proliferated in response to arestricted number of E. tenella 72-II fractions. PBL obtained from16-day old naturally-immunized/challenged birds showed the highest S.I.reactivity to three discrete areas of the immunoblot corresponding toantigens of approximate relative molecular weights (M_(T) S) of 68-75,38-41, and 27-30 kDa (Table 5). Similar parasite proteins of approximateM_(T) S of 25-28 and 38-40 kDa were also identified in crude,concentrated 72 hour antigen by Western using sera from these samenaturally-immunized/challenged birds.

                  TABLE 5                                                         ______________________________________                                        72-II Fraction #                                                                            M.sub.T S Range(kDa)                                                                      S.I. Value                                          ______________________________________                                        2              97-150     1.2                                                 3             75-97       1.2                                                 4             68-75       2.3                                                 5             55-68       1.1                                                 6             48-55       1.5                                                 7             41-48       1.3                                                 8             38-41       1.9                                                 9             35-38       1.3                                                 10            30-35       1.3                                                 11            27-30       2.5                                                 12            22-26       1.2                                                 ______________________________________                                    

F. TNF Assay

Mouse L929 cells ATCC! were suspended in McCoys 5A/5% fetal calf serum(FCS) to 4×10⁵ cells/ml and 0.1 ml added to flat-bottomed microliterplates. After overnight incubation at 37° C., 5% CO₂, log₂ dilutions ofmouse recombinant tumor necrosis factor (TNF) standard Genzyme! (2 μg/mlinitial concentration) or test supernatants were prepared in media inthe absence or presence of actinomycin D (2 μg/ml) and added induplicate to the appropriate wells. Following incubation for 48 hours at37° C., 5% CO₂, plates were washed 1× in Dulbecco's Phosphate-BufferedSaline (DPBS) and cells fixed for 10 minutes at room temperature inmethanol/acetic acid (3:1). Plates were stained for 10 minutes with 0.5%crystal violet/20% methanol and rinsed several times in dH₂ O. Afterwashing, 0.1 ml/well acetic acid (33%) was added and plates allowed tomix on an orbital shaker until stain was uniformly distributedthroughout wells. Absorbance of wells at 600 nm was determined in aMolecular Devices V_(max) automated microplate reader. Data is recordedas % cytotoxicity according the formula:

    % cytotoxicity.sub.dil =A.sub.cont -A.sub.dil /A.sub.cont

where % cytotoxicity_(dil) represents the amount of cell destruction ata given dilution, A_(cont) represents absorbance in control wells (mediaalone) and A_(dil) represents absorbance at a given dilution of testsupernatant. Titer was defined as the reciprocal of the dilutionnecessary to achieve 50% cell cytotoxicity.

                  TABLE 6                                                         ______________________________________                                        Summary of Results from TNF Assay                                             Treatment                                                                             Age of Bird                                                                              Stimulant   Cytotoxic Activity••               ______________________________________                                        5 × 500•                                                                  10         mrz         +                                              5 × 500                                                                         25         mrz         +                                                                 72 h        -                                                                 conconavalin A                                                                            -                                              UI      25         mrz         +                                                                 72 h        -                                                                 conconavalin A                                                                            -                                              ______________________________________                                         •5 × 500 designates doses of 500 oocysts per day for 5 days.      ••(+) designates cytotoxic activity was present but reciproca     titer could not be determined.                                           

G. Interleukin 2 Assay

IL2 responder cells were isolated from the spleens of naive 2-4 week oldB³⁰ B³⁰ birds. Single cell suspensions were adjusted to 5×10⁶ cells/mlin serum-free cLMH containing 2.5 μg/ml Con A and incubated at 40.5° C.,5% CO₂ in T-75 flasks. After 48 hours, nonadherent cells were treatedwith 50 mM α-MM for 20 minutes at 40.5° C. and blast cells isolated bycentrifugation over Histopaque 1077. Viable cells were resuspended inserum-free cLMH/100 mM α-MM to 2×10⁶ /ml and added to round-bottomedmicroliter plates (0.1 ml/well.) Log₂ dilutions of laboratory standardIL2-containing conditioned media were added in quadruplicate toappropriate wells, and serves as a positive control. Serum-free cLMH(negative control) or test supernatant were then added (0.1 ml, 25% v/vfinal well concentration) in quadruplicate and plates incubated at 40°C., 5% CO₂ for 48 hours. Cultures were then pulsed with 1 μCi/well (0.05ml) ³ H!-thymidine for an additional 6 hours. Cells were harvested andcounted as previously described. High and low cpm for each sample werediscarded. Supernatants considered positive for IL2 are those with meancpm values at least two-fold that of serum-free control media from eachplate.

H. Secondary In Vitro Antibody Assay (SIBA)

Log₂ dilutions of antigen (SPZ, mrz, or cell culture antigens) wereprepared in 10 mM borate buffer, pH 9.0, at an initial concentration of1 μg PSP/ml and 0.1 ml/well added to Nunc Immuno-Maixsorb ELISA plates.After overnight incubation at 4° C., wells were blocked using PBS/0.05%Tween 20 (PBS-T) containing 5% skim milk (0.2 ml/well) for 2 hours at37° C. Plates were washed 3× in complete HBSS, 25 mM Hepes, pH 7.4, 1×antibiotic/antimycotic (CHBSS) and sterilized by UV-irradiation for atleast 20 minutes under a sterile hood. PBL or spleen cells were adjustedto 2×10⁷ cells/ml and 0.2 ml added to the first column. Log₂ dilutionsof cells were then made across the entire plate in serum-free cLMH(excluding the last column for each plated antigen) to complete thecheckerboard titration. Wells were brought up to a final volume of 0.2ml using a cLMH and plates incubated for 3 to 5 days at 40.5° C., 5%CO₂. After incubation, plates were vigorously washed 3× using coldPBS-T.

The last column for each plated antigen was incubated with 0.1 ml of E.tenella hyperimmune chicken sera (1:2000 in PBS-T/0.05% BSA) for 1 hourat 37° C. After 3× washes in PBS-T, biotinylated goat anti-chicken IgG(1:2000 in PBS-T/2% skim milk) was added to all wells and incubationcontinued for 1 hour at 37° C. Following 3× washes in PBS-T, wells weretreated with horseradish-peroxidase labelled streptavidin (1:1000 inPBS-T/2% skim milk) for an additional 1 hour. Plates were thoroughlywashed in PBS-T and bound enzyme detected using TMB/peroxidasesubstrate. The enzymatic reaction was stopped after 15 minutes by theaddition of 1M HCl, and the optical density measured at 450 nm in aMolecular Devices V_(max) automated microplate reader.

                  TABLE 7                                                         ______________________________________                                        Secondary In-Vitro B Cell Assay                                               Treatment Group Antigen OD at 450 nm                                          ______________________________________                                        5 × 500   72 h    1.567                                                                 UI      0.910                                                                 mrz     1.750                                                 UI              72 h    0.920                                                                 UI      0.592                                                                 mrz     0.689                                                 ______________________________________                                    

I. Parasite Inhibition Assay (PIA)

The QT-35 cell line (QT35 was provided as a gift from the Department ofVeterinary Services, College of Agriculture, Pennsylvania StateUniversity), grown in Opti-MEM/1% FBS, was seeded at 1×10⁴ cells/well in96-well flat bottom plates. Following an overnight incubation at 40° C.,5% CO₂, cells were pretreated with duplicate log₂ dilutions of positivecontrol conditioned media or test supernatant. One row was pretreatedwith media alone. Following pretreatment for 24 hours, fresh dilutionsof test supernatant were added to cells along with 1×10⁵ E. tenellasporozoites and 1 μCiwell³ H!-uracil. Cultures were incubated for anadditional 24 hours, harvested, and counted as described above. A testsupernatant was considered positive when a 1:8 dilution caused a 30%reduction in mean cpm compared to untreated controls (media alone).

                  TABLE 8                                                         ______________________________________                                        Parasite Inhibition Assay                                                              Cell      Age of  Antigen                                            Treatment                                                                              Source    Bird    Stimulation                                                                             % Inhibition                             ______________________________________                                        NE       LPL       15 days 72 h      17%                                                                 No antigen                                                                              47                                       NE/C     LPL       15 days 72 h      40%                                                                 No antigen                                                                              54%                                      UI/C     LPL       15 days 72 h       7%                                                                 No antigen                                                                              43%                                      Assay controls:                                                               UI       Spleen    21 days conA      54%                                      --       --        --      media      0%                                                                 alone                                              ______________________________________                                         LPL = Lamina Propria Lymphocytes                                              NE = Naturally Exposed (500 ooysts given daily for 5 days)               

Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. For example, use of otherappropriate avian pathogens is expected to produce antigens similar tothe coccidia antigens described herein. Thus vaccines to pathogens otherthan coccidia may be designed using the teachings of the aboveinvention. Such modifications and alterations to the compositions andprocesses of the present invention are believed to be encompassed in thescope of the claims appended hereto.

What is claimed is:
 1. A vaccine against coccidiosis, comprising a cellculture component collected from a culture of cells of cell lineSB-CEV-1\P (ATCC CRL 10497) or a cell line derived therefrom which isable to replicate the prepatent life cycle of an Eimeria avian parasite,which cells are infected with a selected Eimeria avian parasite, whereinthe cell culture component is selected from the group consisting of awhole cell culture preparation, conditioned media which providessignificant weight gain protection or significant improved feedconversion, intact infected host cells, disrupted infected host cells,or a combination thereof, and further comprising an oil in wateradjuvant.
 2. The vaccine of claim 1, wherein the cell line is SB-CEV-1\P(ATCC CRL 10497).
 3. The vaccine of claim 1, wherein the cell linederived from SB-CEV-1\P is is SB-CEV-1\F7 (ATCC CRL 10495).
 4. Thevaccine of claim 1, wherein the cell line derived from SB-CEV-1\P is isSB-CEV-1\G7 (ATCC CRL 10496).
 5. The vaccine of claim 1, wherein theparasite is Eimeria tenella.
 6. A method for vaccinating poultry againstinfection by parasites causing coccidiosis, comprising administering topoultry an effective dose of a vaccine comprising a cell culturecomponent collected from a culture of cells of cell line SB-CEV-1\P(ATCC CRL 10497) or a cell line derived therefrom which is able ofreplicate the prepatent life cycle of an Eimeria avian parasite, whichcells are infected with a selected Eimeria avian parasite, wherein thecell culture component is selected from the group consisting of a wholecell culture preparation, conditioned media which provides significantweight gain protection or significant improved feed conversion, intactinfected host cells, disrupted infected host cells, or a combinationthereof, and further comprising an oil in water adjuvant.
 7. The methodof claim 6, wherein the cell line is SB-CEV-1\P (ATCC CRL 10497).
 8. Themethod of claim 6, wherein the cell line is SB-CEV-1\F7 (ATCC CRL10495).
 9. The method of claim 6, wherein the cell line is SB-CEV-1\G7(ATCC CRL 10496).
 10. The method of claim 6, wherein the parasite isEimeria tenella.